Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells.docVIP

Virology Journal BioMedCentral Methodology Open Access Cloning of the canine RNA polymerase I promoter and establishment of reverse genetics for influenza A and B in MDCK cells Zhaoti Wang and Gregory M Duke* Address: MedImmune, 297 North Bernardo Avenue, Mountain View, CA 94043, USA Email: Zhaoti Wang - wangz@; Gregory M Duke* - dukeg@ * Corresponding author Published: 23 October 2007 Received: 13 September 2007 Accepted: 23 October 2007 Virology Journal 2007, 4:102 doi:10.1186/1743-422X-4-102 This article is available from: /content/4/1/102 ? 2007 Wang and Duke; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Recent incidents where highly pathogenic influenza A H5N1 viruses have spread from avian species into humans have prompted the development of cell-based production of influenza vaccines as an alternative to or replacement of current egg-based production. Madin- Darby canine kidney (MDCK) cells are the primary cell-substrate candidate for influenza virus production but an efficient system for the direct rescue of influenza virus from cloned influenza cDNAs in MDCK cells did not exist. The objective of this study was to develop a highly efficient method for direct rescue of influenza virus in MDCK cells. Results: The eight-plasmid DNA transfection system for the rescue of influenza virus from cloned influenza cDNAs was adapted such that virus can be generated directly from MDCK cells. This was accomplished by cloning the canine RNA polymerase I (pol I) promoter from MDCK cells and exchanging it for the human RNA pol I promoter in the eight plasmid rescue system. The adapted system retains bi-directional transcription of the viral cDNA